Project Summary/Abstract: Asthma is the most common chronic illness of childhood and imposes a profound clinical and financial burden on American children, costing the US health care system ~$50.1 billion dollars annually. The pathogenesis of pediatric asthma is complex, secondary to both genetic predisposition and environmental/infectious exposures. Multiple well-powered genetic association studies (GWAS) and cis- expression quantitative trait loci studies (cis-eQTL) have linked over-expression of the gene ORMDL3 to pediatric-onset asthma. Unfortunately, the role of this gene in asthma pathogenesis remains unclear. Multiple studies have shown ORMDL proteins regulate critical aspects of sphingolipid metabolism, but the underlying immune mechanisms dysregulated by ORMDL3 risk variants are currently undefined. This gap in our basic knowledge impedes the rational targeting of novel therapies to this important new asthma pathway. I have recently performed preliminary studies that have shown ORMDL3 over-expression in dendritic cells (DCs) reduced basal cellular ceramide levels and enhances pro-inflammatory cytokine secretion. I hypothesize that ORMDL3 over-expression in these cells may contribute to the gene's role in asthma pathogenesis. The primary scientific goals of my proposed research project are 1) to establish the cellular and molecular mechanisms linking ORMDL3 over-expression in DCs to pro-inflammatory responses in the lung and 2) to establish the effects of ORMDL3 risk variants on sphingolipid-regulated immune responses in human DCs. To achieve these goals, I will acquire additional training in biostatistics, lipidomic analysis and advanced immunology with the long-term goal of establishing a laboratory focused on the role of sphingolipids in pulmonary inflammation. In the attached proposal, I have outlined an integrated set of multidisciplinary studies to evaluate the role of ORMDL3 in DCs during asthma pathogenesis in both mouse and human systems. In Aim 1, I will determine the in vitro and in vivo effects of ORDML3 over-expression on DC immune function. We will utilize a transgenic ORMDL3 over-expressing mouse model I have developed to probe the functional effects of ORMDL3 on DC antigen processing, T cell activation and DC migration. In Aim 2, we will identify the regulatory effects of ORMDL3 over-expression in DCs by examining the gene's effects on cellular lipids and gene expression during DC maturation. We will utilize both shotgun and targeted lipidomics in combination with RNA sequencing to identify key gene and lipid metabolite pathways that are altered by ORMDL3, leading to enhanced DC-mediated inflammation. In Aim 3, we will determine whether ORMDL3 risk alleles regulate sphingolipid levels in human monocyte-derived DC in ORMDL3 genotyped subjects. Together these studies will directly address the gap in our knowledge regarding sphingolipids and DC function and are predicted to offer insights into the susceptibility mechanisms of pediatric asthma.